DE2018058B2 - BIOTECHNICAL PROCESS FOR THE PRODUCTION OF GLUCOSE ISOMERASE - Google Patents

Description

Temperature and pH ratios, as US Pat. No. 2,950,228 describes a

characterized in that one as a method for converting dextrose to layulose

Microorganism Streptomycss olivaceus NRRL iq (glucose in fructose) with HMe of microorganisms,

3583 uses Pseudomonas hydro-

phila and in example 4 a special Bacillus strain to be named. The Bacillus used in Example 4

Tribe is not specified and therefore is not

15 to be regarded as belonging to the state of the art. To the other examples are used by the

The invention relates to a method for producing dextrose only 11 to 33%, but according to the invention more of glucose isomerase is converted into fructose by aerobic cultivation of one than 40%
Microorganism of the genus Streptomyces in one The British patent specification 1103 394 relates to the xylose-containing nutrient medium with ao conversion of glucose into fructose customary for this purpose with the aid of temperature and pH-value ratios. Microorganisms of the genus Streptomyces. Sweet syrups are widespread and are z. B. is the strain Streptomyces albus YT-No. 4 not to be regarded as state-of-the-art in baking, in the manufacture of confectionery, as it was not stored and used in the beverage industry. This has been. With Streptomyces flavovirens, Strepto-syrups generally consist of sucrose (cane myces echinatur and Streptomyces achromogenus sugar) or dextrose-containing products, which are obtained according to Table 1 conversions of starch hydrolysis, as the main - at most 20, 24 or 36% scored while the sweeteners. If a syrup is required, the conversion of the fructose with the aid of the invention is sweeter than that obtained from sucrose according to the method is at least 40%,
an invert sugar syrup is used. These 30 In Japanese Patent 7,428-66 are produced by acid hydrolysis of sucrose. Production of glucose isomerase is of various types, a mixture of approximately 50% glucose from Streptomyces being specified. A deposited strain (dextrose) and approximately 50% fructose (levulose) is not given. This arises from Table 1 (Example 2). While glucose is slightly less sweet than the patent shows that with Streptomyces Sa sucrose, the fructose is sweeter than sucrose so the best results were obtained from vovirens. From the fact that the total sweetness, compared with sucrose, to the test report (see below) shows that under takes. It is known that dextrose can be converted to fructose under alkaline conditions set out in Example 2 of this Japanese patent specification. This conversion is of great potential importance 40 NRRL 3583 or the strain of St. flavovirens, which is used for the production of sweet syrup. The alkaline conversion, rather at Northern Utilization Research and Develop- ment, however, has had no commercial success ment Division, Agricultural Research Service of the, since the alkaline reaction to an un- United States Department of Agriculture, Peoria, desires high content of ashes in which arise - Illinois, which is available under the designation B 2685, leads to the syrup, which can only be removed uneconomically. 45 Trap of St. olivaceus NRRL 3583 is much higher. The syrup cannot be used unless glucose isomerase activities can be obtained without these ashes being removed. th.

An attempt was then made to convert glucose enzymatically into fructose, also in Japanese Patent 7,430-66. Various Streptomyces strains and species for the production of glucose isomerase in the US Pat. Japanese patents 7,428-66,7 430-66,7 832-66, the tables given there, show that 13 799-66 and 17 640-66 are known that micro- best results with Streptomyces achromogenes organisms from Pseudomonas hydrophilia, Bacillus. A deposited strain is not ange-Streptomyces, such as Streptomyces flavovirens, Streptomyces. As can be seen from the test report (see below) Hervormyces echinatur, Streptomyces achromogenus, Streptomyces, better results are grown with the aid of the myces albus and the like used according to the invention, as well as Lactobacillus fermenti in the strain Streptomyces olivaceus NRRL 3583 in a suitable nutrient medium obtained than with the strepto can, whereby enzymes are formed, the strain belonging to Glucose myces achromogenus, which has isomerase properties. However, these well-known Northern Utilization Research and Development Divi processes did not establish themselves commercially, 60 sion, Agricultural Research Service of the United, because either the yields of enzyme during the States Department of Agriculture, Peoria, Illinois, with the enzyme production process or the yields of the Designation B 2120 is available when both are grown in fructose, when the enzyme is used to convert xylose-containing nutrient media. Glucose used were too low. As can be seen from the tables of the Japanese patent specification The invention relates to a process for the preparation of 65 7430-66, xylose-containing media of glucose isomerase by aerobic cultivation give better results than media which do not contain xylose from a microorganism of the genus Streptomyces.
Xylose-containing nutrient medium in the case of the customary for this purpose. In Japanese patent specification 17 640-66 there are 7

^ωεβ ! ^even - <& ² ^ isomerase can be used. Optionally, the

Si ⁸ U ^ge ^^ ^{et Sini Eia ZeUen} obtained by potion and then by loading

is not specified. Are broken Tn the local _known methods. The

^ ÄL ^ nJ ^^ £ ^ ^ Streptomyces standing broken cells and their released ones

phaeochromogenes replaced. As shown in the experiment 5 cell contents can be used as a source for glucose isomerase

is based (see below), a stored one leads to be used. Besides, the cells can too

Representatives of Stoptomyces phaeochromogenus under z. B. by ultrasound or homogenization apparatus

Applying appropriate nutrient media at a lower rate and the debris can get through

good results than the centrifugation according to the invention can be removed. The protruding

^S u- ^ Tv.Ai, », a ■ " ^ liquid can be used directly as a source for the glucose iso-

u% S> S ^ L ^na ? Japanese patent merase can be used or it can be converted into a fine sniff 7832-66 Streptomyces bobdiae. Powder can be lyophilized for later use. A deposited trunk is not specified. A comparison The enzyme may, however, leads to the supernatant by known Ausfäll- ^T l * · Λ * ^Γ Γ ^obe P8 ^enamite ^ Japanese Patent 15 medium, such as methanol or ammonium sulfate from the above-described violators of Streptomyces bobihae, schnft precipitated 7 430 will produce less good results than there
used strain of Streptomyces achrcmogenes. The activity of the glucose iso-

Japanese patent specification 13 799-66 relates to the merase for fructose production.

Production of fructose from glucose can be determined using both methods of determination, production of Lactobacillus fermenti. _20th

There it is stated that the glucose isomerase of the determination method 1

Lactobacillus fermenti-Mycels under certain conditions a 50% (weight / volume) aqueous

conditions after 72 hours of incubation a glucose solution of glucose with 0.005 M ammonium chloride,

Conversion reached before1 48%. According to the inven- 0.1m magnesium sulfate and 0.001m cobalt chloride

According to the method, however, a glucose-Um- a _{5 is} produced. This solution had a pH of

Conversion to fructose m level of at least 40% 9.0. 5 ml of the buffered glucose specified above

achieved within 1 hour. ] ö _SU ng were treated with 5 ml of a glucose isomerase

The solution suitable for the process according to the invention is mixed. This last-mentioned solution

Strain belongs to Streptomyces olivaceus. He became a suspension of whole bacterial cells, a can

at Northern Utilization Research and Develop- 30 suspension of broken cells or the end

ment Division, Agricultural Research Service of the enzyme solution that

United States Department of Agriculture, Peoria. The above mentioned

Illinois, deposited and assigned number NRRL 3583. Mix was shaken well and kept at 70 ° C for 60 minutes

This culture is warmed up to the public indefinitely. 1 ml of the resulting reaction mixture

available. ₃₅ was taken away and 9 ml of O, 5N-Perchlor-

The Streptomyces olivaceus NRRL 3583 is acid mixed. Further inclines of agar were held with water and dilutions can be made in a xylose, as they were necessary to cultivate the holding nutrient medium. Cheaper - to produce a suitable final color. To 1 ml of this, the medium also contains other charcoal mixture was added 0.2 ml of 1.5 weight hydrates, a nitrogen source and inorganic salts. 40 percent solution of cysteine hydrochloride added Typical sources of carbohydrates are corn starch, Dex. 6 ml of a mixture of trose, lactose, milk solids, wheat bran and the like were then added to this. Typically 190 ml of distilled water and 450 ml of concentrated nitrogen sources are peptones, yeast extract, sulfuric acid. Immediately thereafter, meat extract, amino acids and the like were added. Typical inorganic 0.2 ml of a 0.12 percent strength by weight alcoholic salts are sodium chloride, magnesium sulfate and the like 45 solution of carbazole. The entire Ge-These carbohydrates, nitrogen sources and inorganic was then shaken and 30 minutes in case of seeing salts are known. The growth in the 40 ⁰ C allowed to stand. The optical density of the xylose used can then be measured in purified form, standing purple in color, or in the form of a crude xylose-containing material using a light source with a wavelength of. 50 560 nm was used. The measured values thus obtained

The Streptomyces olivaceus NRRL 3583 is green for the optical density were based on the values

compared to a standard curve on which the fructose

tation is plotted for about 20 to about 50 hours at concentration versus optical density

Temperature was overall from about 15 to about 35 ^0C. The percentage of glucose that is in fructose

breeds. 55 had been converted at temperatures below about 15 ° C, it was stated that

and at temperatures above approximately 35 ^° C.

the yield of the desired enzyme fairly determination method 2

low. The preferred growth temperature is 62.5% (w / v) aqueous

about 25 to about 35 ° C. It is conveniently made solution of glucose by taking under

Atmospheric pressure applied but pressures above 60 stir slowly 625 g of anhydrous glucose to 300 ml

and below atmospheric pressure can be added without hot distilled water. The solution was

particular advantages or disadvantages are applied. cooled to room temperature and were used for this

The initial pH of the growth medium should be 125 ml of a 1 M aqueous solution of Tris- (hydroxy-

be between about 6.8 and 7.1. methyl) aminomethane of pH 8.5, 125 ml of a

The glucose isomerase according to the invention is in 65 1 M aqueous solution of magnesium sulfate and 50 ml Formed inside the bacterial cells, which during their production of a 0.025 M aqueous solution of cobalt chloride grow. The cells can be set from the fermentation. The resulting mixture was then with Broth filtered off and diluted to 11 directly as a source for the glucose- distilled water.

10 ml of the above glucose solution was centrifuged at 15,000 rpm for 20 minutes, and the

placed in a 25 ml volumetric flask. There was an optical rotation (in degrees) of the protruding

sufficient amount of the enzyme to be examined draws * liquid was determined by known methods,

give to a reduction of the specific rota- It was a blank sample in the same way, but

tion of about 2.9 to about 7.5 degrees, 5 without the enzyme. The optical rotation of the

as will be described later, the contents of the flask were also determined as blank. The specific

then diluted to 25 ml with distilled water and rotation was [χ] Ή of the sample or the blank sample

the resulting mixture was 2 hours "" at 60 ⁹ e 2 "or twice the value observed optir

ditched. The reaction was then seen by watching rotation. The percentage conversion of

Set of 1 ml of a 0.5 M aqueous solution of perio glucose in fructose was given by the following formula

chloric acid canceled. The mixture was then calculated ^:

Percent Conversion =, _JQQ

This determination method was also used, conditions in 1 minute 1 μΜοΙ glucose in fructose to convert the activity in glucose isomerase units to ao. Calculate the enzyme activity in glucose iseanerase, one glucose isomerase unit becoming the units (GIE) of the enzyme sample examined Amount of enzyme means that among those given is calculated according to the following formula:

GIE / g or OIE / 1 = ^ fructose formed x 0.0463

mg or ml of enzyme used

where the ug of fructose formed by multiplying fermentation was continued for 24 hours at 32 ° C, the value calculated above for the percentage. The fermentation broth was authorized at 40,000 rpm centri-glucose conversion with a factor of 6.25 · IG ^{8 in} order to separate off the bacterial cells. The cells was reckoning. were then frozen and lyophilized. The activity The invention is illustrated by the following Examples 35 of the resulting enzyme powder. Tuning method 2 determined. There was one

Activity of 42.6% conversion of glucose to

Example 1 fructose.

A culture of Streptomyces olivaceus NRRL 40 B e i s ρ i e 1 3

3583 was placed in a 250 ml flask containing the

100 ml of an aqueous mixture with Γ, 0% xylose, a culture of Streptomyces olivaceus NRRL 3583

1.0% peptone, 0.50% meat extract, 0.25% yeast was placed in a fermentation vessel, which was equipped as in the extract, 0.50% sodium chloride and 0.05% Magne- Example 2 and 101 of the example 2 contained siumsulfat with a pH of 6.8 to 7.0. 45 described medium contained. All the percentages given above are stirred at 400 rpm and air is given in an amount of weight / volume. The one with the bacterium passed 2 l / minute. The fermentation was inoculated flask contents were then continued on a back and for 48 hours at 21 to 31 ⁰ C. The Ferment reciprocating shaker tationszellen cm with a rash of 5 were separated in a homo- and 176 motions per minute for 24 hours at 50 genisator disrupted, and the cell debris were 30 C ^p shaken. The bacterial cells were removed from the by centrifugation. The resulting fermentation broth is filtered off. The liquid was lyophilized by the determination and activation method 1 determined a conversion of minority of the powder by determination method 2 at least 40% glucose into fructose. certainly. 320 GIE / g were obtained.

55 The hergei according to the method according to the invention

^ eispjel 2 posed glucose isomerase is used to convert

Glucose in fructose among other things than that for be?

A culture of Streptomyces olivaceus NRRL 3583 suitable for mood conditions. This was done while stirring in a fermentation vessel is described in the following example,
give, the 101 of an aqueous mixture with 0.7% 60
Xylose, 0.3% refined corn starch, 1.0% peptone,

0.5% meat extract, 0.25% yeast extract, 0.50% B P i s ρ 1 e 1 4

Sodium chloride and 0.05% magnesium sulfate and

a pH of 7.0. All of the above- An aqueous QluGose solution containing 25% (weight /

The above percentages relate to weight / volume (65 volume) glucose was used with some bacteria. The mixture was stirred at 400 rpm and air was mixed with terry cells, which corresponded to that in the case; at a rate of 3 volumes of air per game 2 specified method has been grown Volume of the medium passed through per minute. The goods. The activity of the bacterial cells was ent

according to method 2, and the cells were used in an amount of 1.5 GIE per g of glucose in the glucose solution. Allowed the resulting mixture with a pH of 8.0 for 72 hours at 60 ⁰ C to react. The resulting solution was examined according to Method 2 and it was found that a conversion of glucose to fructose of 40.7% had taken place.

Culture

B 2120

B 2208

B 2685

B 3559

NRRL 3583

Test report

Attempts have been made to determine the effectiveness of several known strains of Streptomyces or to compare the enzymes produced by them with the glucose isomerase obtained according to the invention. In addition to the strain Streptomyces olivaceus NRRL 3583 the following with the Northern Utilization F-esearch and Development Division, Agricultural Research Service of the United States Department of Agriculture, Peoria, Illinois examined.

B 2120 Streptomyces achromogenes,

B 2208 Streptomyces albus,

B 2685 Streptomyces flavovirens,

B 3559 Streptomyces phaeochromogenes.

These cultures were all kept on agar under known conditions at room temperature until they had sporulated enough. The cultures were then stored in the refrigerator at 4 ° C.

A. A first growth medium was prepared consisting of an aqueous solution of 1% xylose, 1% polypeptone, 0.3% K _S HPO _4, and 0.1% MgSO ₄ · 7 H ₈ O.

In addition, a first fermentation medium was prepared, consisting of an aqueous solution of 3% wheat bran, 4% corn water, 0.1% MgSO ₄ -7H ₁ O and 0.025% CoCljS-OH ₂ O. The pH was adjusted to 6 with NaOH. 5 set

100 ml of the above growth medium was placed in five individual 250 ml wide neck Erlenmeyer flasks and the flasks and contents were sterilized with 1.4 atm steam for 20 minutes. Portions of the cultures indicated above were then added to the individual flasks and the inoculated Flask incubated for 48 hours at 30 ° C on a rotary shaker at 275 rpm.

100 ml of the fermentation medium specified above were each placed in five individual 250 ml wide-necked Erlenmeyer flasks and the flasks and the contents were sterilized for 20 minutes with steam of 1.4 atmospheres. In each case, portions of the incubated growth medium indicated above were added to the individual flasks in an amount of 1 percent by volume, and the flasks inoculated in this way were ^{incubated for 24 hours at 30 °} C. on a rotary shaker at 270 rpm. Then equal proportions of the individual flask contents were removed and examined. The fermentation was then continued under the conditions given above for a total of 48 hours

The glucose isomerase activity of each fraction of the The resulting bacterial cells were determined by method 2. The following results were obtained.

Glucose isomerase (GIE / 1.) Activity 48 hours 24 hours 0 0 0 0 0 0 79 0 263 0

B. A second fermentation medium was made, consisting of an aqueous solution of 0.5% xylan, 1% polypeptone, 0.3% K ₈ HPO ₄ and 0.1% MgSO ₄ -7 H ₂ O.

Individual components of the growth medium erstei above were inoculated with the five Kulturei and incubated for 48 hours at 3O ⁰ C. Then 100 ml each of the second fermentation

_ ₀ medium put into individual flasks and inoculated with part of the incubated growth medium and incubated for 2A or 48 hours as indicated above. The glucose isomerase activity of the individual sample was determined as indicated above. One received the

_{as the} following results.

Culture

B 2120

B 2208

B 2685

B 3559

NRRL 3583

Glucose isomerase (Gffi / 1.) activity 48 hours 24 hours 346 230 0 0 164 218 0 0 250 113

C. A second growth medium was prepared consisting of an aqueous solution of 1% xylose, 1% peptone, 0.5% meat extract, 0.25% yeast extract, 0.5% NaQ, and 0.05% MgSO ₄ · 7H ₁ O. The pH was adjusted to 7.0 with NaOH

A third fermentation medium was produced, the composition of which corresponded to the second growth medium given above, with the exception that a mixture of 0.7% xylose and 0.3% corn starch was used instead of 1% xylose. The pH was also adjusted to 7.0
Individual proportions of the second given above

- _o growth medium were inoculated with the different cultures and 24 hours at 3O ⁰ C on a rotary shaker at 275 rpm incubated Each 100 ml of the above third fermentation medium were placed in individual piston and nut

_ ₅ 2 percent by volume of the incubated growth medium inoculated and incubated as described above for 24 or 48 hours. The glucose isomerase activity of the individual samples was determined as above

60 Gfacose isomerase
Activities (GIE / 1.)
24 hours | 48 hours 1112
390
1344
1064
1953 B 2120 .. - ..
6 ₅ B2208 ..
B2685 1022
0
1757
892
2779 B3559 NRRL3583

309529/402

D. A fourth fermentation medium was prepared consisting of an aqueous solution of 0.7% xylose, 0.3% corn starch, 0.25% yeast hydrolyzate and 2% corn water. The pH was with Sodium hydroxide adjusted to 7.2.

Individual portions of the fourth fermentation medium specified above were inoculated with the individual cultures and ^{incubated for 24 hours at 30 °} C. on a rotary shaker at 275 rpm. Another 100 ml each of the fermentation medium specified above were added to individual flasks and inoculated with 1 percent by volume of the incubated medium and incubated as described above for 24 or 48 hours. The glucose isomerase activity of the individual samples was determined as described above. The following results were obtained.

Culture

Glucose isomerase (GIE / 1.) activity 48 hours 24 hours 1341 1300 0 0 1456 279 664 1000 3517 2674

B 2120

B 2208

B 2685

B 3559

NRRL 3583

From these test results it can be seen that Streptomyces olivaceus NRRL 3583 is the only one of the Streptomyces strains used here produced glucose isomerase activity in all media used and that in almost all cases a higher glucose isomerase activity could be obtained than with the rest of the tribes.

Claims (1)

1, 2 Temperature and pH value relationships, the Claim: characterized in that one is used as a microorganism Streptomyces olivaceus NRRL 3583 begins. That Process for the production of Glucojse-Iso- enzyme thus formed can be ermerase in good yields by aerobic cultivation of a "microprganis- 5 and has better properties for get out of the genus Streptomyces in a xylose the conversion of glucose into fructose than the bis-containing Nutrient medium with known enzymes customary for this purpose.